10. AAV Gene Therapy for Improved Wound Healing: Insights From Fetal Scarless Repair

PURPOSE: Cutaneous wound healing is a complex process typically characterized by significant inflammation and fibrosis, resulting in formation of a scar. Improper wound healing can lead to complications, including functionally limiting and disfiguring scars. Prior to late gestation, in contrast to adult skin, fetal skin can regenerate with minimal scarring. However, the mechanisms governing scarless healing are not fully understood. This study aims to identify critical components of scarless healing by profiling the fetal transition from scarless to scarring healing. We also investigated whether there are pro-regenerative or pro-fibrotic factors that play essential roles in orchestrating this transition and whether these factors could be manipulated via viral gene therapy vectors to improve healing and fibrosis in adult skin. METHODS: Pregnant rats of gestational age E16.5 (scarless) and E18.5 (scarring) were used. A 1 mm2 section of integument was excised from the dorsal thorax of individual fetuses, with tissue collected at 1 and 3 days post-injury. Single-nuclei RNA sequencing (snRNA-seq) was performed on nuclei isolated from both uninjured and injured fetal skin to identify candidate genes. For viral studies, adult wild-type mice received two full-thickness splinted dorsal skin injuries. Four days post-injury, a recombinant adeno-associated virus (rAAV) vector containing an injury-specific enhancer element, LEN, and an effector gene was administered via local subcutaneous injection. One wound was treated with an rAAV containing interleukin-1 receptor antagonist (IL-1Ra), a known anti-inflammatory and anti-fibrotic factor, while the other received a green fluorescent protein (GFP) control vector. Fetal and adult wound tissue was analyzed histologically and by immunofluorescence to assess inflammation, collagen organization, and scarring. RESULTS: Fetal skin demonstrated the ability to regenerate normal tissue architecture after cutaneous injury, with no scar formation. snRNA-seq identified candidate genes, including POSTN, which encodes a pro-fibrotic factor periostin, and SLIT2, which encodes a pro-regenerative factor slit guidance ligand 2. Scarless tissue exhibited lower POSTN expression and higher SLIT2 expression compared to scarring tissue. Histological analysis showed reduced inflammation and improved collagen organization, with fibers more closely resembling uninjured skin, in IL-1Ra-treated wounds. Gross analysis also revealed an increased healing rate in the first week post-injury compared to controls. CONCLUSION: An exploration into the fetal transition from scarless to scarring healing has the potential for improving wound healing in humans. We show that a LEN-AAV system for viral delivery of genes, including IL-1Ra, allows for targeted expression at sites of injury, improving wound healing. Ongoing research on viral manipulation of wound healing with candidate genes may pave the way for novel gene therapies in scarring and chronic non-healing wounds